Method of treatment of hemorrhagic disease using a factor VIIa/tissue factor inhibitor

ABSTRACT

The present invention provides methods of treating hemorrhagic fevers where selective inhibitors of fVIIa/TF are used as a treatment for hemorrhagic fevers and have therapeutic effects which include ameliorating and/or preventing coagulopathy and inflammatory responses. These inhibitors include certain proteins which are part of a family termed Nematode-Extracted Anticoagulant Proteins (“NAPs”). Other inhibitors include Tissue Factor Pathway Inhibitor (“TFPI”) and TFPI analogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/431,629, filed May 6, 2003 from which priorityis claimed pursuant to 35 U.S.C. §120, which application is herebyincorporated by reference in its entirety.

BACKGROUND AND INTRODUCTION TO THE INVENTION

A number of viruses have been reported to cause lethal hemorrhagicdisease in humans and certain other primates. These viruses includeviruses from a number of viral families, which include Filoviridae,Arenaviridae, Bunyaviridae, and Flaviridae. In particular theFiloviridae, which include the Ebola and Marburg viruses, have resultedin significant morbidity and mortality in infected populations. Atpresent there are no satisfactory disease specific therapies or vaccinesfor diseases caused by the Filoviridae.

Patients affected with hemorrhagic fevers develop a severe consumptivedisseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is typically characterizedby wide-spread systematic activation of the coagulation cascaderesulting in excess thrombin generation. In addition, activation of thefibrinolytic system coupled with the consumption of coagulation factorsresults in a depletion of clotting factors and degradation of plateletmembrane glycoproteins.

Conventional treatment of DIC has been aimed primarily at treatment ofthe underlying etiologic disease process and secondarily at thecoagulopathy that results in the thrombotic and hemorrhagicmanifestations. Reported therapies include replacement therapy ofcoagulation factors by transfusion of fresh frozen plasma. Heparin hasbeen reported as sometimes used in combination with replacement therapy.

Tissue factor (“TF”) is a 47 kDa transmembrane glycoprotein that is themajor cellular trigger of blood coagulation under physiologicconditions. The factor VIIa-tissue factor(“fVII/TF”) catalytic complexis able to generate factor Xa via direct activation of factor X, andindirectly through the activation of factor IX, thus initiating thrombingeneration. It has been reported that tissue factor also plays animportant role in disease processes resulting from the activation of thecoagulation pathway. For example, TF levels are reported to be elevatedduring bacterial sepsis and this is believed to contribute directly tothe pathogenesis of multiple organ failure (Doshi et al., “Evolving roleof tissue factor and its pathway inhibitor”, Crit. Care. Med. (2002)30(5 Suppl): 5241-5250). In addition, a number of viruses have beenreported to activate the coagulation system following infection; suchactivation may also be triggered by the up regulation of TF expression(Bowman, et al., “Procoagulant and inflammatory response ofvirus-infected monocytes”, European J. Clin. Invest. (2002) 32: 759-766;Baugh et al., “Regulation of extrinsic pathway factor Xa formation bytissue factor pathway inhibitor”, J. Biol. Chem. (1998) 273: 4378-4386;Taylor et al., “Active site inhibited factor VIIa (DEGR VIIa) attenuatesthe coagulant and interleukin-6 and -8, but not tumor necrosis factor,responses of the baboon to LD100 Escherichra coli”, Blood (1998) 91:1609-1615). A variety of inflammatory stimuli, including bacterial cellproducts, viral infection and cytokines, have been reported to inducethe expression of TF on the surface of endothelial cells and monocytes,thereby activating the coagulation pathway (Doshi, et al.).

Tissue Factor Pathway Inhibitor (TFPI) is an endogenous systemicallycirculating plasma protein which is said to function as a physiologicalanticoagulant by inhibiting VIIa/TF complexes and preventing theinitiation of coagulation. TFPI contains multiple Kunitz-type proteaseinhibitor domains, and is the principal physiologic inhibitor ofTF/FVIIa. TFPI has been reported to bind to and inhibit factor Xadirectly, prior to forming a quaternary inhibitory complex withTF/FVIIa, thereby inhibiting thrombin generation (Baugh et al.).Processes for preparing recombinant TFPI have been reported. See, e.g.U.S. Pat. No. 6,300,100 to Kamel et al.

In addition to its role in initiating coagulation, the TF/FVIIa has beenreported to have direct pro-inflammatory effects independent of theactivation of coagulation in man (Taylor et al.). In experimentalsettings where animals were depleted of TFPI, the animals were reportedto have a demonstrated sensitivity to bacterial endotoxin and a higherpropensity to develop intravascular coagulation (Doshi et al.). In alethal E. coli sepsis model in baboons, treatment with TFPI was reportedto attenuate the procoagulant and inflammatory cytokine interleukin-6(IL-6) response and to prevent mortality (Creasey et al., “Tissue factorpathway inhibitor reduces mortality from Escherichia coli septic shock”,J. Clin. Invest. (1993) 91(6): 2850-2860). However, in healthy humanvolunteers administered low dose bacterial endotoxin, blocking TF/FVIIawith TFPI was reported to have no impact on inflammatory cytokines, butto completely prevent endotoxin-induced activation of coagulation(deJonge et al., “Tissue factor pathway inhibitor does not influenceinflammatory pathways during human endotoxemia”, J. Infect. Dis. (2001)183(12): 1815-1818). A phase II clinical trial of recombinant TFPI(rTFPI) in patients with severe sepsis, the rTFPI group was reported todemonstrate accelerated decrease of IL-6 plasma levels (Reinhart et al.,“Assessment of the safety of recombinant tissue factor pathway inhibitorin patients with severe sepsis, a multicenter randomized, placebocontrolled single blind, dose escalation study”, Crit. Clin. Med. (2001)29(11): 2081-2089). A phase III trial of trifacogin, a rTFPI, in severesepsis failed to show a reduction in the primary end-point of 28-day allcause mortality (Doshi et al.).

Protein C (PC) is another component of the natural anticoagulant systemin mammals. Unlike TFPI, which acts at the level of TF/fVIIa, the PCpathway is reported to inhibit coagulation by down regulating thrombinformation via the proteolytic inactivation of the non-enzymaticco-factors factor Va and factor VIIIa (Esmon C., “Protein C pathway insepsis”, Annals of Medicine (2002) 34:598-605). The active component ofthe PC pathway is termed activated PC (aPC). aPC is formed by the actionof thrombin bound to the non-enzymatic co-factor thrombomodulin. Arecombinant form of aPC (drotrecogin alfa) has been reported to reducethe incidence death in patients suffering from severe bacterial sepsis(Bernard et al., “Efficacy and safety of recombinant human activatedProtein C for severe sepsis”, N. Engl. J. Med. (2001) 344:699-709).

Published United States Patent Application, publication number US2001/0028880 A1, is said to relate to the treatment of viral hemorrhagicfever with Protein C.

SUMMARY OF THE INVENTION

According to one aspect, the present invention is directed to treatmentof viral hemorrhagic fever by administration of a selective inhibitor ofthe TF/VIIa complex which initiates the activation of coagulation.

In view of the published reports on the effects of inhibiting theTF/VIIa complex by different inhibitors, it is not predictable as towhat effects a particular inhibitor of TF/VIIa will have on theassociated inflammation responses induced by either bacterial or viralinfection. Furthermore, little is known regarding the role of TF inmediating the coagulation and inflammatory responses to viral infectionparticularly hemorrhagic viral infection.

According to one aspect, the present invention is directed to a methodfor treating a mammal having a hemorrhagic fever which comprisesadministering to said mammal an amount of a selective inhibitor ofFactor VIIa/tissue factor (“fVIIa/TF”) effective to ameliorate symptomsof said fever.

According to an alternate aspect, the present invention is directed to amethod of treating a mammal having a hemorrhagic fever which comprisesadministering to said mammal a therapeutically effective amount of acompound which specifically inhibits the catalytic activity of thefVIIa/TF complex in the presence of Factor Xa (“fXa”) or catalyticallyactive fXa derivative (including the zymogen Factor X (“fX”)) and doesnot specifically inhibit the catalytic activity of the fVIIa/TF complexin the absence of fXa or catalytically inactive fXa derivative and doesnot specifically inhibit the activity of Factor VIIa (“fVIIa”) in theabsence of tissue factor (“TF”) and does not specifically inhibitprothombinase.

In an additional aspect, the present invention is directed to a methodof treating coagulopathy and/or an inflammatory response due tohemorrhagic fever in a mammal which comprises administering to saidmammal an amount of a selective fVIIa/TF inhibitor effective to decreaseor prevent said coagulopathy and/or inflammatory response in saidmammal.

The hemorrhagic fever to be treated may be caused by a virus. Suchhemorrhagic fevers may be caused by viruses such as the Filoviridae,Arenaviridae Bunyaviridae and Flaviridae. The methods of the presentinvention may be particularly suited to treat viral hemorrhagic feverscaused by the Filoviridae.

Suitable fVIIa/TF inhibitors and compounds which inhibit the catalyticactivity of the fVIIa/TF complex include members of the family ofNematode-Extracted Anti-coagulant Proteins (NAPs”) having fVIIa/TFinhibitory activity, tissue factor pathway inhibitor (“TFPI”) andanalogs of tissue factor pathway inhibitor.

Suitable NAPs for use in the methods of the present invention include anisolated protein having Factor VIIa/TF inhibitory activity and havingone or more NAP domains, wherein each NAP domain includes the sequence:Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10,wherein

(a) A1 is an amino acid sequence of 7 to 8 amino acid residues;

(b) A2 is an amino acid sequence;

(c) A3 is an amino acid sequence of 3 amino acid residues;

(d) A4 is an amino acid sequence;

(e) A5 is an amino acid sequence of 3 to 4 amino acid residues;

(f) A6 is an amino acid sequence;

(g) A7 is an amino acid residue;

(h) A8 is an amino acid sequence of 11 to 12 amino acid residues;

(i) A9 is an amino acid sequence of 5 to 7 amino acid residues; and

A10 is an amino acid sequence; wherein each of A2, A4, A6 and A10 has anindependently selected number of independently selected amino acidresidues and each sequence is selected such that each NAP domain has intotal less than about 120 amino acid residues.

Certain suitable NAPs for use according to the methods of the presentinvention include AcaNAPc2 (SEQ. ID. NO. 3) and AcaNAPc2/proline (SEQ.ID. NO. 4). Also suitable for use according to the methods of thepresent invention are TFPI and TFPI analogs.

Definitions

The terms “Factor Xa” or “fXa” or “FXa” are synonymous and are commonlyknown to mean a serine protease within the blood coagulation cascade ofenzymes that functions to form the enzyme thrombin as part of theprothrombinase complex.

The term “catalytically inactive fXa derivative” includes the zymogenFactor X (“fX” or “FX”), as well as other catalytically inactive fXaderivatives.

The phrase “Factor Xa inhibitory activity” means an activity thatinhibits the catalytic activity of fXa toward its substrate.

The phrase “Factor Xa selective inhibitory activity” means inhibitoryactivity that is selective toward Factor Xa compared to other relatedenzymes, such as other serine proteases.

The phrase “Factor Xa inhibitor” is a compound having Factor Xainhibitory activity.

The terms “Factor VIIa/Tissue Factor” or “Tissue Factor/Factor VIIa” or“fVIIa/TF” or “FVIIa/TF” or “TF/fVIIa” are synonymous and are commonlyknown to mean a catalytically active complex of the serine proteasecoagulation factor VIIa (fVIIa) and the non-enzymatic protein TissueFactor (TF), wherein the complex is assembled on the surface of aphospholipid membrane of defined composition.

The phrase “fVIIa/TF inhibitory activity” or “TF/fVIIa inhibitoryactivity” means an activity that inhibits the catalytic activity of thefVIIa/TF complex in the presence of fXa or catalytically inactive fXaderivative (including the zymogen fX). The phrase “fVIIa/TF selectiveinhibitory activity” means inhibitory activity that is selective towardfVIIa/TF compared to other related enzymes, such as other serineproteases, including FVIIa and fXa.

The phrase a “fVIIa/TF inhibitor” is a compound having fVIIa/TFinhibitory activity in the presence of fXa or catalytically inactive fXaderivatives.

The phrase “serine protease” is commonly known to mean an enzyme with acommon three-dimensional fold, comprising a triad of the amino acidshistidine, aspartic acid and serine, that catalytically cleaves an amidebond, wherein the serine residue within the triad is involved in acovalent manner in the catalytic cleavage. Serine proteases are renderedcatalytically inactive by covalent modification of the serine residuewithin the catalytic triad by diisopropylflourophosphate (DFP).

The phrase “serine protease inhibitory activity” means an activity thatinhibits the catalytic activity of a serine protease.

The phrase “serine protease selective inhibitory activity” meansinhibitory activity that is selective toward one serine proteasecompared to other serine proteases.

The phrase “serine protease inhibitor” is a compound having serineprotease inhibitory activity.

The term “prothrombinase” is commonly known to mean a catalyticallyactive complex of the serine protease coagulation Factor Xa (fXa) andthe non-enzymatic protein Factor Va (fVa), wherein the complex isassembled on the surface of a phospholipid membrane of definedcomposition.

The phrase “anticoagulant activity” means an activity that inhibits theclotting of blood, which includes the clotting of plasma.

The terms “selective,” “selectivity,” and permutations thereof, whenreferring to activity of a compound or composition toward a certainenzyme, mean that the compound or composition inhibits the specifiedenzyme with at least 10-fold higher potency that it inhibits other,related enzymes. Thus, the activity of the compound or composition isselective toward that specified enzyme.

The term “substantially the same” when used to refer to proteins, aminoacid sequences, cDNAs, nucleotide sequences, and the like refer toproteins, cDNAs or sequences having at least about 80% homology with theother protein, cDNA, or sequence.

The terms “AcaNAPc2” and rNAPc2” refer to a recombinant protein of theNAP family. The preparation and sequence of AcaNAPc2 is described inU.S. Pat. No. 5,866,542.

The terms “AcaNAPc2/proline,” “AcaNAPc2P,” “rNAPc2/proline” and“rNAPc2/Pro” refer to a recombinant protein having the amino acidsequence of AcaNAPc2 which has been modified to add a proline residue tothe C-terminus of the sequence of AcaNAPc2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the expression vector pYAM7sp8/rNAPc2 used in theexpression of recombinant AcaNAPc2/proline.

DETAILED DESCRIPTION OF THE INVENTION

A. Inhibitors of fVIIa/TF

A number of selective inhibitors of fVIIa/TF have been reported.According to one aspect of the present invention, these inhibitors areused as a treatment for hemorrhagic fevers and have therapeutic effectswhich include ameliorating and/or preventing coagulopathy andinflammatory responses. These inhibitors include certain proteins whichare part of a family termed Nematode-Extracted Anticoagulant Proteins(“NAPs”). Other inhibitors include Tissue Factor Pathway Inhibitor(“TFPI”) and TFPI analogs.

1. NAP Proteins

U.S. Pat. No. 5,866,542 to Vlasuk et al. describes a family of proteinstermed “Nematode-Extracted Anticoagulant Proteins (“NAPs”), one class ofwhich is described as having Factor VIIa/TF inhibitory activity. Thisclass of proteins include an isolated protein having Factor VIIa/TFinhibitory activity and having one or more NAP domains, wherein each NAPdomain includes the sequence:Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10(“FORMULA III”), wherein

(a) A1 is an amino acid sequence of 7 to 8 amino acid residues;

(b) A2 is an amino acid sequence;

(c) A3 is an amino acid sequence of 3 amino acid residues;

(d) A4 is an amino acid sequence;

(e) A5 is an amino acid sequence of 3 to 4 amino acid residues;

(f) A6 is an amino acid sequence;

(g) A7 is an amino acid residue;

(h) A8 is an amino acid sequence of 11 to 12 amino acid residues;

(i) A9 is an amino acid sequence of 5 to 7 amino acid residues; and

(j) A10 is an amino acid sequence; wherein each of A2, A4, A6 and A10has an independently selected number of independently selected aminoacid residues and each sequence is selected such that each NAP domainhas in total less than about 120 amino acid residues.

Suitable NAP proteins within this aspect of the invention have at leastone NAP domain. Preferred are NAPs having one or two NAP domains.Preferred are proteins having at least one NAP domain substantially thesame as the NAP domain of AcaNAPc2 (SEQ. ID. No. 3). The NAP proteinAcaNAPc2 (SEQ. ID. NO. 3) has one NAP domain and is a preferred NAPaccording to this aspect of the invention. Also preferred isAcaNAPc2/proline (SEQ. ID. NO. 4). AcaNAPc2/proline is a recombinantprotein having the sequence of AcaNAPc2 and which has an additionalproline residue at the C terminus.

Preferred NAP proteins include those in which A2 is an amino acidsequence of 3 to 5 amino acid residues, A4 is an amino acid sequence of6 to 19 amino acid residues, A6 is an amino acid sequence of 3 to 5amino acid residues, and A10 is an amino acid sequence of 5 to 25 aminoacid residues.

In certain preferred NAPs, A3 has the sequence Asp-A3_(a)-A3_(b),wherein A3_(a) and A³ _(b) are independently selected amino acidresidues. More preferably, A3 is Asp-Lys-Lys.

In certain preferred NAPs, A4 is an amino acid sequence having a netanionic charge.

Also preferred are NAPS where A5 has the sequenceA5_(a)-A5_(b)-A5_(c)-A5_(d) wherein A5_(a) through A5_(d) areindependently selected amino acid residues. Preferably, A5_(a) is Leuand A5_(c) is Arg.

In certain preferred NAPs, a preferred A7 amino acid residue is Val orIle, more preferably Val.

Certain preferred NAPs include those in which A8 includes the amino acidsequence A8_(a)-A8_(b)-A8_(c)-A8_(d)-A8_(e)-A8_(f)-A8_(g)

wherein

(a) A8_(a) is the first amino acid residue in A8,

(b) at least one of A8_(a) and A8_(b) is selected from the groupconsisting of Glu or Asp, and

(c) A8_(c) through A⁸ _(g) are independently selected amino acidresidues.

Preferably, A8_(c) is Gly, A8_(d) is selected from the group consistingof Phe, Tyr, and Leu, A8_(e) is Tyr, A8_(f) is Arg, and A⁸ _(g) isselected from Asp and Asn. A preferredA8_(c)-A8_(d)-A8_(e)-A8_(f)-A8_(g) sequence is Gly-Phe-Tyr-Arg-Asn (SEQ.ID. NO. 5).

In one embodiment, the present invention is directed to methods of usinga suitable NAP molecule having factor VIIa/tissue factor inhibitoryactivity wherein

(a) A3 has the sequences Asp-A3_(a)-A3_(b), wherein A3_(a) and A3_(b)are independently selected amino acid residues;

(b) A4 is an amino acid sequence having a net anionic charge;

(c) A5 has the sequence A5_(a)-A5_(b)-A5_(c)-A5_(d), wherein A5_(a)through A5_(d) are independently selected amino acid residues, and

(d) A7 is selected from the group consisting of Val and Ile. Use ofpharmaceutical compositions comprising one or more of these NAP proteinshaving factor VIIa/tissue factor inhibitory activity and apharmaceutically acceptable carrier are contemplated by this embodiment.NAP proteins used according to the methods of the present invention haveat least one NAP domain. Preferred are NAPs having one or two NAPdomains. The NAP proteins AcaNAPc2 and AcaNAPc2/proline have one NAPdomain and are preferred NAPs according to this embodiment of theinvention.

In another preferred embodiment, a suitable NAP molecule for use in themethods of the present invention is one wherein

(a) A3 is Asp-Lys-Lys;

(b) A4 is an amino acid sequence having a net anionic charge;

(c) A5 has the sequence A5_(a)-A5_(b)-A5_(c)-A5_(d) wherein A5_(a)through A5_(d) are independently selected amino acid residues;

(d) A7 is Val;

(e) A8 includes an amino acid sequence A8_(a)-A8_(b)-Gly-Phe-Tyr-Arg-Asn(SEQ. ID. NO. 6), wherein at least one of A8_(a) and A8_(b) is Glu orAsp. Use of pharmaceutical compositions comprising such NAP proteins anda pharmaceutically acceptable carrier also are contemplated by thisinvention. These NAP proteins have at least one NAP domain. Preferredare NAPs having one or two NAP domains. The NAP proteins AcaNAPc2 (SEQ.ID. NO. 3) and AcaNAPc2/proline (SEQ. ID. NO. 4) each have one NAPdomain and are preferred NAPs according to this embodiment of theinvention.

Certain preferred NAP proteins having Factor VIIa/TF inhibitory activityas described above are derived from a nematode species. A preferrednematode species is selected from the group consisting of Ancylostomacaninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Necatoramericanus, and Heligomosomoides polygyrus. These NAPs may convenientlybe prepared by recombinant means. Particularly preferred are the NAPproteins AcaNAPc2 and AcaNAPc2/proline. AcaNAPc2 was derived fromAncylostoma caninum using recombinant methods.

U.S. Pat. No. 5,866,542 describes the preparation of recombinant NAPproteins, including AcaNAPc2 and AcaNAPc2/proline.

2. Tissue Factor Pathway Inhibitor

U.S. Pat. No. 6,300,100 describes Tissue Factor Pathway Inhibitor andprocess for its preparation by recombinant methods.

U.S. Pat. No. 5,378,614 describes the preparation of certain analogs ofTFPI.

B. Selection of Compounds Having Factor VIIa/Tissue Factor InhibitoryActivity

The fVIIa/TF inhibitory activity of NAPs and other compounds usedaccording to the methods of the present invention can be determinedusing protocols described herein. Example 3 describes fVIIa/TF assays.There, the fVIIa/TF-mediated cleavage and liberation of the tritiatedactivation peptide from radiolabeled human factor IX (³H-FIX) or theamidolytic hydrolysis of a chromogenic peptidyl substrate are measured.Interestingly, certain fVIIa/TF inhibitors require the presence of fXaor catalytically inactive fXa derivative in order to be active fVIIa/TFinhibitors. However, certain NAP and other fVIIa/TF inhibitors wereequally effective in the presence of fXa in which the active site hadbeen irreversibly occupied with the peptidyl chloromethyl ketoneH-Glu-Gly-Arg-CMK (EGR), and thereby rendered catalytically inactive(EGR-fXa). While not wishing to be bound by any one explanation, itappears that these proteins having fVIIa/TF inhibitory activity form abinary complex with fXa by binding to a specific recognition site on theenzyme that is distinct from the primary recognition sites, P₄-P-₁,within the catalytic center of the enzyme. This is followed by theformation of a quaternary inhibitory complex with the fVIIa/TF complex.Consistent with this hypothesis is that EGR-fXa can fully support theinhibition of fVIIa/TF by these compounds which are inhibitory forfVIIa/TF despite covalent occupancy of the primary recognition sites(P₄-P₁) within the catalytic site of fXa by the tripeptidyl-chloromethylketone (EGR-CMK).

The fVIIa/TF inhibitory activity of these compounds also can bedetermined using the protocol in Example 7, as well as the fXa assaysdescribed above, and in Examples 5 and 6. There, the ability of acompound to inhibit the catalytic activity of a variety of enzymes ismeasured and compared to its inhibitory activity toward the fVIIa/TFcomplex.

Certain of these compounds specifically inhibit the catalytic activityof the fVIIa/TF complex in the presence of fXa or catalytically inactivefXa derivative (including the zymogen Factor X), but do not specificallyinhibit prothrombinase. Preferred compounds according to this aspect ofthe invention have the characteristics described above for an isolatedprotein having Factor VIIa/TF inhibitory activity and having one or moreNAP domains. A preferred protein according to this aspect of theinvention is AcaNAPc2 or AcaNAPc2/Pro.

These compounds are identified by their fVIIa/TF inhibitory activity inthe presence of fXa or a fXa derivative, whether the derivative iscatalytically active or not. The protocol in Example 4 can detect acompound's inactivity toward free fXa or prothrombinase. Data generatedusing the protocols in Example 3 will identify compounds that requireeither catalytically active or inactive fXa to inhibit fVIIa/TF complex.

C. Administration and Formulation

According to one aspect of the present invention, the fVIIa/TF inhibitoris administered to a patient in need of treatment as soon as thehemorrhagic fever is detected. It may be preferred to treat the patientat as early a stage in the hemorrhagic fever as possible, preferably ator before onset of symptoms. Thus, with respect to hemorrhagicfever-causing viruses it may be advantageous to administer the fVIIa/TFprior to exposure to the virus or as soon after exposure as possible.

The compounds or pharmaceutical compositions thereof used according tothe methods of the present invention are administered in vivo,ordinarily in a mammal, preferably in a human. In employing them invivo, the compounds and pharmaceutical compositions can be administeredto a mammal in a variety of ways, including orally, parenterally,intravenously, subcutaneously, intramuscularly, colonically, rectally,nasally or intraperitoneally, employing a variety of dosage forms.Administration is preferably parenteral, such as intravenous on a dailybasis. Alternatively, for some compounds or pharmaceutical compositionsadministration is preferably oral, such as by tablets, capsules orelixirs taken on a daily basis.

In practicing the methods of the present invention, the compounds orpharmaceutical compositions used according to the methods of the presentinvention are administered alone or in combination with one another, orin combination with other therapeutic or in vivo diagnostic agents.

As is apparent to one skilled in the medical art, a therapeuticallyeffective amount of the compounds or pharmaceutical compositions of thepresent invention will vary depending upon the age, weight and mammalianspecies treated, the particular compounds employed, the particular modeof administration and the desired effects and the therapeuticindication. Because these factors and their relationship to determiningthis amount are well known in the medical arts, the determination oftherapeutically effective dosage levels, the amount necessary to achievethe desired result of ameliorating the effects caused by the hemorrhagicfever will be within the ambit of one skilled in these arts.

Typically, administration of the compounds or pharmaceutical compositionof the present invention is commenced at lower dosage levels, withdosage levels being increased until the desired therapeutic effect isachieved which would define a therapeutically effective amount. For thecompounds of the present invention, alone or as part of a pharmaceuticalcomposition, such doses are between about 0.001 mg/kg and 100 mg/kg bodyweight, preferably between about 0.05 and 10 mg/kg, body weight.

To assist in understanding, the present invention will now be furtherillustrated by the following examples. These examples as they relate tothis invention should not, of course be construed as specificallylimiting the invention and such variations of the invention, now knownor later developed, which would be within the purview of one skilled inthe art are considered to fall within the scope of the invention asdescribed herein and hereinafter claimed.

EXAMPLE 1

Production of Recombinant Nematode-Extracted Anticoagulant Proteinc2/Proline (AcaNAPc2/Proline or rNAPc2/Pro)

A. Preparation of rNAPc2/Pro Expression Plasmid and Pichia pastorisExpressing Clone

The rNAPc2/Pro gene was cloned into the P. Pastoris expression vector,pYAM7sp8¹, using PCR rescue. The pYAM7sp8 vector (see, FIG. 1) is aderivative of pHIL-D2². It contains the promoter and transcriptionaltermination signal of the Pichia pastoris AOX1 gene, a secretion signalpeptide (a fusion of the Pichia pastoris acid phosphatase signalsequence and the pro sequence of a hybrid S. cerevisiae α-matingfactor), and the HIS4 marker for selecting transfectants.

The PCR primers used to rescue the rNAPc2 gene from the phage clone³were:

(SEQ. ID. NO. 1) A8 (^(5′)GCG TTT AAA GCA ACG ATG CAG TGT GGT G^(3′))and (SEQ. ID. NO. 2) A9 (^(5′)C GCT CTA GAA GCT TCA TGG GTT TCG AGT TCCGGG ATA TAT AAA GTC^(3′)).

These primers add Dral and Xbal sites to the 5′ and 3′ ends of therescued DNA fragment, respectively. Primer A9 also inserts a prolinecodon just before the termination codon.

FIG. 1 depicts a rNAPc2/Pro P. pastoris expression vector.

The resulting PCR fragment was digested with Dral and XbaI and clonedinto pYAM7sp8 digested with Stul and Spel. Ligating the blunt ends ofpYAM7sp8 (Stul) and the PCR fragment (Dral) resulted in an in-framefusion of the P. pastoris secretion signal peptide to the mature portionof rNAPc2/Pro. Ligating the XbaI and Spel ends of the PCR fragment andpYAM7sp8 resulted in the destruction of the pYAM7sp8 Spel site. The P.pastoris expression strain was constructed by integrating the expressioncassette into the P. pastoris genome by homologous recombination. ThepYAM7sp8/NAPc2/Pro construct was digested with Notl. The digestedplasmid was electroporated into P. pastoris GS115 (his4-) cells.Transfectants were screened for methanol utilization phenotype (mut+)and high-level expression of rNAPc2/Pro. A single isolate (designated asGS115/AcaNAPc2P-55) was selected for generation of the Master Cell Bank(MCB). The production strain was analyzed by Southern blots that wereprobed by radiolabeled rNAPc2/Pro or HIS4 genes. These blots showed thatmultiple copies of the expression cassette were integrated at the3′-site of the AOX1 gene.

i. Master Cell Bank (MCB)

The Master Cell Bank (MCB) was prepared using a prebank of a singlecolony isolate (GS115/AcaNAPc2P-55). The flask containing YEPD flaskmedium (bacto peptone, yeast extract, and dextrose) with 2% glucose wasinoculated with 0.5 mL of the prebank and grown to an optical density(A_(550 nm)) of 0.5 to 1.0. The culture was harvested, diluted withglycerol to a final concentration of 15% as a cryopreservative, andfrozen in cryovials stored below −60° C.

ii. Manufacturer's Working Cell Bank

A Manufacturer's Working Cell Bank (MWCB) was manufactured from a vialof the MCB. A vial of the MCB was used to inoculate a flask containingYeast Peptone medium (peptone and yeast extract) and 2% dextrose. Theflask was incubated at 28±2° C. and 250 rpm until the optical density(A_(600 nm)) was 17.0±5.0. The culture was harvested, and thecryopreservative, glycerol, was added to a final concentration of 9%.Aliquots of 1.1±0.1 mL were filled into 2.0 mL cryovials, frozen andstored at −70±10° C.

iii. Test Methods Used for Analysis of Master Cell Bank

Host Identification: The rNAPc2/Pro cell bank culture was streaked ontoTrypticase Soy Agar (TSA) plates and the plates were incubated forgrowth. The isolate was set up for identification using the Vitek®identification system which utilizes a temperature controlled chamberand photometric sensor unit to monitor changes in turbidity of theisolate suspension which has been inoculated into a Vitek® yeast testcard containing substrates for 26 conventional biochemical tests. Forthe rNAPc2/Pro cell bank host identifications, the resulting reactionbiopattern was compared to a positive control organism (Pichia pastoris,ATCC No. 76273) reaction biopattern.

Viable Cell Concentration: Viable cell concentration of the rNAPc2/Procell bank was measured by enumeration of viable colony forming units(CFU) by preparation of serial dilutions from three cell bank vials (oneeach from beginning, middle, and end). The dilutions were plated intriplicate onto TSA plates and incubated CFUs are counted andcalculations performed to determine cell concentration as CFU/mL.

Structural Gene Sequence Analysis: The cell bank culture was preparedfor gene sequencing by amplifying the rNAPc2/Pro gene incorporated intothe host genome using the Polymerase Chain Reaction (PCR) technique. ThePCR product was purified and the concentration determined. The PCRproduct was then sequenced using dideoxy chain termination (Sanger)method. The resulting gene sequence of the cell bank was compared to theknown DNA sequence of rNAPc2/Pro. Identity was confirmed by a 100%match.

Non-Host Contamination Assay: The rNAPc2/Pro fermentation broth wastested for non-host contamination by inoculating 100 mL onto each ofnine TSA plates. Three plates were incubated at three temperatures (20to 25° C., 30 to 34° C., and 35 to 39° C.). During the seven dayincubation period the plates were inspected for microbial colonies thatdiffer from the characteristic host, particularly noting differences incolony morphology, color and/or colony size. A Gram stain was alsoperformed on the final read plate. Appropriate negative controls wereincluded in the assay.

iv. Test Methods Used for Analysis of Manufacturer's Working Cell Bank

Host Identification: The rNAPc2/Pro cell bank culture was streaked ontoSabouraud Dextrose Agar (SDA) plates and the plates were incubated forgrowth at 20 to 25° C. for 7 days. In parallel, a positive control (ATCCstrain of K. pastoris, an alternate name for P. pastoris) was streakedonto SDA plates in the same manner. Selected colonies that grew werethen tested by gram staining.

After the incubation, at least two morphologically similar colonies fromeach SDA plate were selected from the test sample SDA plates andpositive control SDA plates. These colonies were subcultured ontoseparate SDA plates and incubated at 20 to 25° C. for 7 days. The API20C AUX test and Gram staining was then performed on growth from eachsubculture plate. The API test system (bioMérieux SA; Marcy l'Etoile,France) was a manual microbial identification test that contained 20miniature biochemical tests. The 20C AUX test strip contained 20biochemical tests specific for identification of yeast. The API testingresults for the rNAPc2/Pro cell bank test sample were compared to theresults obtained for the positive control to confirm identification.

Viable Cell Concentration: Viable cell concentration of the rNAPc2/Procell bank was measured by enumeration of viable colony forming units(CFUs) by preparation of serial dilutions from two cell bank vials, onevial pulled before freezing the bank and one vial pulled after the bankwas frozen. A 100 μL aliquot of each dilution was plated onto duplicateTSA and incubated for 5 to 7 days. All plates with countable colonies(30 to 300 CFUs) were counted. Counts obtained from plates of the sametest sample dilution were averaged, multiplied by that dilution anddivided by the 100 μL aliquot size to report results as CFU/mL.

DNA Sequencing: Total DNA was isolated from the newly created cell bank(test article). The NAPc2/Pro gene was amplified by polymerase chainreaction (PCR) using primers homologous to the 5′ and 3′ sequences ofthe cloned NAPc2/Pro gene. The resulting DNA fragment (approximately 500bp) was purified using standard methods and used as a template for DNAsequencing using a primer walking strategy performed using the ThermoSequenase radiolabeled terminator cycle sequencing kit (AmershamBiosciences, Piscataway, N.J.). The sequencing films were read bydigitization and the sequence data was assembled and analyzed usingSequencher™ software, version 3.0 (Gene Codes Corp., Ann Arbor, Mich.).The consensus sequence produced from the test article was then comparedto the theoretical sequence for the NAPc2/Pro gene.

Non-host Contamination: Prior to freezing the newly created cell bank(test article), a vial was submitted for non-host testing. A sample ofthe broth was diluted one-thousand fold in saline. Duplicate plates ofnine different media types were inoculated with 100 μL of the dilutedtest sample. In addition, a positive control (ATCC strain of K.pastoris, an alternate name for P. pastoris) was diluted and inoculatedonto plates in the same manner. Another set of plates was not inoculatedand designated as the negative controls plates. All plates except SDAwere incubated at 30 to 35° C. for 48 to 72 hours; the SDA plates wereincubated at 20 to 25° C. for 7 days. The plates were examined forgrowth after 1, and 2 or 3 days. In addition, the SDA plates wereexamined for growth after 7 days. Any aberrant colonies were identifiedby API testing and Gram stain.

After day 2 or 3, at least two morphologically similar colonies fromeach TSA plate were selected from the test sample TSA plates andpositive control TSA plates. These colonies were subcultured ontoseparate TSA plates and incubated at 30 to 35° C. for 48 to 72 hours.The API 20C AUX test and gram staining was then performed on growth fromeach subculture plate. The API test system (BioMérieux SA; Marcyl'Etoile, France) was a manual microbial identification test thatcontained 20 miniature biochemical tests. The 20C AUX test stripcontained 20 biochemical tests specific for identification of yeast. TheAPI testing results for the test article were compared to the resultsobtained for the positive control to confirm identification.

B. Production of rNAPc2/Pro

The production of the rNAPc2/Pro consists of fermentation, recovery,purification, bulk filtration and fill. The following sections describethe individual unit operations for each stage of the process.

This section describes the fermentation procedures for production ofrNAPc2/Pro. The rNAPc2/Pro protein is produced as a secreted protein byP. pastoris. The fermentation process for rNAPc2/Pro consists of seedflasks, a seed fermentation, and a production fermentation.

i. Seed Fermentation

The purpose of the seed fermentation is to provide a suitably denseinoculum for the production fermentation. Three vials of the MWCB werethawed and one milliliter was used to aseptically inoculate each ofthree two-liter baffled shake flasks containing 250 mL of autoclavedmedium at a pH of 6.0±0.1 (Table I). The flasks were covered andtransferred to an incubator shaker at 250±5 rpm and 28±2° C. The flaskswere incubated for a period of 27.5±2.0 hours and until the celldensity, as measured by Wet Cell Weight, was ≦30 g/L. Once these twoparameters were achieved, the contents of two of the flasks wereaseptically transferred into an autoclaved inoculum bottle.

TABLE I Seed Flask Medium Components Concentration Potassium Phosphate,dibasic 2.30 g/L Potassium Phosphate, monobasic 11.8 g/L Glycerol 10mL/L Yeast Nitrogen Base without amino acids 13.4 g/L Biotin 0.4 mg/L

The medium for the seed fermentation (Table III) was transferred into aseed fermentor. The medium was steam sterilized, allowed to cool, andthe pH was adjusted to 5.0±0.2 with filter-sterilized 28-30% ammoniumhydroxide. Filter-sterilized antifoam solution of 5% (v/v) KFO880 in 50%methanol was then added through a septum to a concentration of 0.5 mL/L.When the temperature stabilizes at 28.0±1.0° C., the medium wasinoculated with the contents of the seed flask inoculum bottle at aratio of 2.5%. The culture pH in the fermentor was maintained at 5.0±0.2with 28-30% ammonium hydroxide. The growth of the fermentation wasmonitored by measuring the Wet Cell Weight.

TABLE II PTM4 Trace salts Components Concentration Cupric Sulfate,Pentahydrate 2.0 g/L Sodium Iodide 0.08 g/L Sodium Molybdate, Dihydrate0.2 g/L Zinc Chloride 7.0 g/L Ferrous Sulfate, Heptahydrate 22.0 g/LBoric Acid 0.02 g/L Cobalt Chloride, Hexahydrate 0.5 g/L ManganeseSulfate, Monohydrate 3.0 g/L d-Biotin 0.2 g/L Sulfuric Acid 1.0 mL/L

TABLE III Seed Fermentation Medium Components Concentration Phosphoricacid, 85% 8.8 mL/L Calcium Sulfate, Dihydrate 0.93 g/L MagnesiumSulfate, Heptahydrate 14.3 g/L Potassium Hydroxide 4.2 g/L AmmoniumSulfate 5.0 g/L Potassium Sulfate 18.2 g/L Glycerol, 100% 7.9 mL/L PTM4Trace Salts (see Table II) 3.0 mL/L

The fermentation was conducted for 15±2 hours and to a final wet cellweight of ≦20 g/L. A portion of the seed fermentation culture wastransferred through a steam-sterilized transfer line into an autoclavedinoculum can. A sample of the final seed fermentation was tested forNon-Host Contamination.

ii. Production Fermentation

The purpose of the production fermentation is to produce high levels ofrNAPc2/Pro protein. To achieve this, the culture was grown to a highcell density prior to rNAPc2/Pro gene induction. The medium for theproduction fermentation (Table IV) was prepared in a productionfermentor. These media components were dissolved and mixed with purifiedwater USP and then steam sterilized. The tank was cooled to its initialoperating temperature of 28.0±1.0° C. A filter-sterilized antifoamsolution of 5% (v/v) KFO880 in 50% methanol was then added. The pH wasadjusted to its initial operating range of 5.0±0.3 withfilter-sterilized 28-30% ammonium hydroxide. When the initial operatingconditions were achieved, the medium was inoculated with the contents ofthe seed fermentation inoculum can at a ratio of 1 kg inoculum per 10 kgof initially batched medium (pre-inoculation weight). The productionfermentation consists of four distinct phases: glycerol batch, glycerolfed-batch, methanol adaptation, and methanol induction. Throughout thefermentation, the dissolved oxygen levels were maintained atapproximately 35% by the addition of air at a constant rate and the useof backpressure and variable agitation. If additional oxygen is neededonce the maximum agitation is achieved, the air stream is supplementedwith oxygen gas. The pH of the culture in the fermentor was maintainedwith 28 to 30% ammonium hydroxide. The antifoam solution wasperiodically added to control foaming.

TABLE IV Production Fermentation Medium Components ConcentrationPhosphoric acid, 85% 8.8 mL/L Calcium Sulfate, Dihydrate 0.93 g/LMagnesium Sulfate, Heptahydrate 14.3 g/L Potassium hydroxide 4.13 g/LPotassium Sulfate 18.2 g/L Ammonium Sulfate 5.0 g/L Glycerol, 100% 23.8mL/L PTM4 Salts (see Table II) 3.0 mL/L

The first phase of the fermentation, the glycerol batch phase, buildsbiomass. The fermentor was run at 28±2° C. until the glycerol in themedia was depleted, as detected by an oxygen spike caused by the ceaseof metabolism of the glycerol. This was followed by the glycerolfed-batch phase in which a 50% w/w glycerol solution was fed to theculture at 18.0±1.0 mL/kg pre-inoculation weight/hour for a total of 8.5hours to increase biomass and repress expression. During the first 4.5hours of this glycerol feed phase, the pH set point of the culture waslowered from 5.0±0.3 to 2.9±0.1 in 0.5 pH units each hour. Temperaturewas maintained at 28±2° C. throughout this phase. The WCW was ≦225 g/Lprior to the end of the glycerol fed-batch phase.

In the methanol adaptation phase, the glycerol feed was terminated andreplaced with a methanol feed which induced the organism to producerNAPc2/Pro. The methanol feed (containing 6.0 mL/L KFO880 antifoam) wasstarted at 3.0 mL/kg pre-inoculation weight/hour. The culture was testedfor methanol adaptation beginning at 2 hours after initiating themethanol addition. The test for methanol adaptation consisted of brieflyterminating the feed and verifying a spike in dissolved oxygen. Afterthe first four hours of methanol addition the temperature was lowered to25±1° C. over a 2 hour period. After the first four hours of methanoladdition and after verification that the culture was utilizing methanol,the methanol feed rate was increased by 1.0 mL/kg pre-inoculationweight/hour. Methanol consumption was measured hourly to ensure that themethanol was being completely depleted, at which point the methanol feedrate was increased by 1.0 mL/kg pre-inoculation weight/hour, up to afinal feeding rate of 6.0 mL/kg pre-inoculation weight/hour.

During the methanol induction phase, the processing conditions at theend of the methanol adaptation phase were maintained throughout theremaining fermentation. Beginning at approximately 48 hours of totalfermentation time, the production of rNAPc2/Pro was monitored bydetermining the concentration of the broth supernatant, as measured byC8 Reversed-Phase assay. The production fermentor was harvested after144 to 168 hours in the production fermentor, and after the rNAPc2concentration as measured by the C8 Reversed-Phase assay was ≦0.55 g/L.A sample of the final fermentation was tested for Non-HostContamination.

C. Purification of rNAPc2/Pro

i. Recovery Step

The purpose of the Recovery step is to separate the rNAPc2/Pro from thecell debris and to exchange the product into a buffer suitable for thefirst purification chromatography step. The medium used to achieve theseparation is an expanded bed ion exchange chromatography column ofStreamline SP XL resin (Amersham Biosciences). The fermentation brothwas diluted with purified water until the conductivity was ≦9 mS/cm. Thesolution was adjusted to a concentration of 150 mM acetate and the pHwas adjusted to pH 3.1±0.2 using 17.4 M acetic acid. The load solutionwas applied to an expanded resin bed that has been equilibrated with 500mM sodium acetate, pH 3.2 followed by 50 mM sodium acetate, pH 3.2. Thecolumn was washed in upflow mode with 50 mM sodium acetate, pH 3.2 andthen with 50 mM sodium acetate/150 mM NaCl, pH 3.2. The resin bed wasallowed to settle and an additional wash was performed using the 50 mMsodium acetate/150 mM NaCl, pH 3.2 in downflow mode. rNAPc2/Pro waseluted by the application of 50 mM sodium acetate/350 mM NaCl, pH 3.2and the rNAPc2/Pro concentration was measured by the C8 Reversed-Phaseassay.

In preparation for the Source 15PHE chromatography step, solid sodiumsulfate was added to the Streamline eluate to a final concentration of0.85 M. The pH was adjusted to 3.1±0.2 using 2.4 M citric acid and theconductivity was verified to be 100±10 mS/cm. The conditioned Streamlineeluate was filtered through 0.45 μm filters.

Following the Recovery step, the initial purification step partiallypurifies the product by removing some protein and non-proteinaceouscontaminants from rNAPc2/Pro using a column of Source 15PHE hydrophobicinteraction chromatography (HIC) media (Amersham Biosciences). Thefiltered, conditioned Streamline eluate was applied to a Source 15PHEcolumn previously equilibrated with 50 mM sodium citrate/1.1 M sodiumsulfate, pH 3.0. After loading, the column was washed with theequilibration buffer. The rNAPc2/Pro protein was eluted from the columnusing a 15 column volume gradient from 1.1 M to 0.3 M sodium sulfate in50 mM sodium citrate, pH 3.0, followed by a gradient hold of the 0.3 Msodium sulfate until the UV absorbance returned to baseline. Fractionswere collected across the rNAPc2/Pro elution peak and then analyzed bythe C18 Reversed-Phase assay. Those fractions containing a high purityof rNAPc2/Pro were pooled and tested by the Concentration by UV assay.The pH of the Source 15PHE pool was adjusted to pH 5.3±0.1 by theaddition of 5N NaOH.

The purpose of ultrafiltration/diafiltration procedure 1 (“UF/DF#1”)following the HIC procedure is to concentrate the product and toexchange the rNAPc2/Pro into the buffer used for the Source 15Q (HIC)chromatography. Regenerated cellulose ultrafiltration filters of a 3 kDmolecular weight pore size were utilized. The pH-adjusted Source 15PHEpool was concentrated to 2.0±0.5 g/L (as measured by Concentration byUV) on the membranes of UF/DF#1 that were previously equilibrated with50 mM sodium acetate, pH 5.3. The pool was then diafiltered with ≦5volumes of 50 mM sodium acetate, pH 5.3, and until the pH was 5.3±0.1and the conductivity was ≦6.0 mS/cm. The diafiltered UF/DF#1 pool wasfiltered through a 0.2 μm filter in preparation for loading onto theSource 15Q column.

The final chromatography unit operation (“Final UF/DF”) removed most ofthe remaining protein and non-proteinaceous contaminants from rNAPc2/Prousing a column of Source 15Q ion exchange chromatography media (AmershamBiosciences). The filtered UF/DF#1 pool was applied to the Source 15Qchromatography column previously equilibrated with 500 mM sodiumacetate, pH 5.3 followed by 50 mM sodium acetate, pH 5.3. After loading,the column was washed with the 50 mM sodium acetate, pH 5.3equilibration buffer. A 20 column volume linear gradient from 0 to 400mM NaCl in 50 mM sodium acetate, pH 5.3 was applied to the column.Fractions are collected across the elution peak and analyzed by the C18Reversed-Phase assay. Those fractions of high rNAPc2/Pro purity werepooled and tested by the Concentration by UV assay.

The purpose of the Final UF/DF is to concentrate the product and toexchange the rNAPc2/Pro into the final formulation buffer. Regeneratedcellulose ultrafiltration filters of a 3 kD molecular weight pore sizeare utilized. The Source 15Q pool was concentrated to 12.0±0.5 g/L (asmeasured by Concentration by UV) on the Final UF/DF membranes that havebeen previously equilibrated with the final formulation buffer, 65 mMsodium phosphate/80 mM sodium chloride, pH 7.0. The pool was thendiafiltered with ≦6 volumes of the formulation buffer, and until the pHwas 7.0±0.1.

The purified rNAPc2/Pro was transferred into a Class 100 area andfiltered using a Millipak 0.2 μm filter into autoclaved 1 liter moldedNalgene Tefzel® FEP (fluorinated ethylene propylene) 1600 series bottlewith a molded, linerless, non-contaminating Tefzel®ETFE (ethylenetetrafluoroethylene copolymer) screw closure. The bottles weretransferred into a −20±10° C. freezer for storage. This material may beformulated using either the standard liquid or lyophilized formationssuitable for treating patients.

ii. Description of rNAPc2/Pro In-Process Test Methods

a. Wet Cell Weight

Approximately 1.5 mL of fermentation samples were added to taredmicrocentifuge tubes and centrifuged at 10,000 rpm for approximately 5minutes. The supernatant from each tube was decanted and the tubescontaining the solids were weighed. The wet cell weight is equal to thenet weight divided by the original sample volume.

b. Non-Host Contamination Assay

The final broth of the seed and production fermentation samples weretested for non-host contamination by inoculating 100 μL onto each ofnine TSA plates. Three plates were incubated at three temperatures (20to 25° C., 30 to 34° C. and 35 to 39° C.). During the seven dayincubation period the plates were inspected for microbial colonies thatdiffer from the characteristic host, particularly noting differences incolony morphology, color and/or colony size. A Gram stain was alsoperformed on the final read date. Appropriate negative controls wereincluded in the assay.

c. C8 Reversed-Phase Assay (Concentration and Purity)

The supernatants of the production fermentation samples, and StreamlineSP XL samples were 0.22 μm filtered and then injected onto a KromasilC8, 4.6×250 mm Reversed-Phase column. The column was equilibrated with22% acetonitrile, 0.1% trifluoroacetic acid (TFA) prior to the sampleinjection. A linear gradient was then run from 22-28% acetonitrile in0.1% TFA over twenty minutes at 1 mL/min to elute the rNAPc2/Promaterial. rNAPc2/Pro standard dilutions of known concentrations wereused to generate a standard curve based upon a linear regression ofrNAPc2/Pro mg/mL versus peak area. The amount of rNAPc2/Pro in anysample is extrapolated from the standard curve and divided by the volumeof sample injected to determine the concentration of rNAPc2/Pro in thesamples. rNAPc2/Pro purity is calculated as a percent of the total peakarea.

d. Concentration by UV

The concentration of each purification pool from the Source 15PHEthrough the UF/DF-Final was determined using its absorbance at 280 nm ona suitably calibrated spectrophotometer. The instrument was blankedusing the applicable buffer solution prior to running the test samples.Test samples were prepared in triplicate by diluting within the linearrange (between 0.13 to 1.62 AU). The average absorbance at 280 nm wasdivided by the extinction coefficient [0.59 AU*cm⁻¹*(mg/mL)⁻¹] andmultiplied by the dilution factor to obtain the concentration in mg/mL.

e. C18 Reversed-Phase Assay (Purity)

The purity of the Source 15PHE fractions and pool, UF/DF #1 pool, Source15Q fractions and pool, and the Final UF/DF pool were each analyzed bythe C18 Reversed-Phase assay. rNAPc2/Pro was separated from othercomponents of a sample by linear gradient Reversed-Phase chromatography.Samples were diluted, if necessary, to approximately 1 mg/mL in cPBS and30 μL was injected into a Waters Symmetry C18 Reversed-Phase column (5μm particles, 4.6 mm I.D.×250 mm length) equilibrated in 78% mobilephase A (0.1% TFA in water) and 22% mobile phase B (0.1% TFA inacetonitrile). The percentage of mobile phase B was then increasedlinearly to 26% over a twenty minute time period, using a 1 mL/min flowrate. The peaks were monitored by the UV detector at 210 nm. The purityof rNAPc2/Pro is calculated by dividing the area of the rNAPc2/Pro peakby the total peak area in the chromatogram and expressing that as apercentage.

iii. Characterization of rNAPc2/Pro

The identity of rNAPc2/Pro was confirmed using amino acid sequenceanalysis and peptide mapping techniques. The purity of the preparationwas judged to be >96% using C18 reverse-phase chromatography.

iv. Bioactivity

rNAPc2/Pro prolongs the clotting time of human plasma initiated by theaddition of thromboplastin in a concentration-dependent manner. Theanticoagulant effect of rNAPc2/Pro on the clotting of human plasma isdirectly measured in an automated Prothrombin Time (PT) Clotting Assayusing rabbit brain thromboplastin (tissue factor, Simplastin-Excel) toinitiate clotting.

Both rNAPc2/Pro reference standard and rNAPc2/Pro sample were diluted to1035 nM in assay buffer. The test instrument (Coag-A-Mate MAX,BioMérieux) then made a set of dilutions in human plasma from thestarting preparation and measured the resulting clotting time (CTs) inseconds. Curves were defined by linear regression fit of the log CTs ofthe rNAPc2/Pro versus the dilution concentrations. The bioactivity ofthe test article was then calculated as the ratio of the slope of thecurve of the test article to the curve of the reference standard timesthe activity of the reference standard.

REFERENCES

1. Laroche Y, Storme V, De Meutter J, Messens J, Lauwereys M. (1994)Biotechnology 12, 1119-1124.

2. Despreaux C W, Manning R F. (1993) Gene 106, 35-41.

3. Jespers L S, Messens J H, De Keyser A, Eckhout E, Van Den Brande I,Gansemans Y G, Lauwereys M J, Vlasuk G P, Stanssens P E. (1995)Biotechnology 13, 378-382.

EXAMPLE 2

Evaluation of Factor VIIa/Tissue Factor (fVIIa/TF) Inhibitor in aNon-Human Primate Model of Acute-Infection with Ebola Virus (EBOV).

Filovirus-naïve rhesus macaques (Macaca mulatta) were randomized intoone experimental group consisting of four monkeys (three females, onemale), and one control group consisting of one animal (male). Allanimals were exposed to 1000 plaque forming units (PFU) of EBOV (Zairesubtype) by intramuscular injection following a ≦ one week acclimationto a biosafety level (BSL)-4 animal room located at the United StatesArmy Medical Research Institute of Infectious Diseases (USAMRIID), FortDetrick, Md. Animals in the experimental group were treated with 30μg/kilogram body weight (kg) of the test fVIIa/TF inhibitor(rAcaNAPc2/proline) by a single subcutaneous injection within 1 hourafter virus challenge, and continuing daily for 14 days after exposureto EBOV. The control animal received an equivalent volume of placebo (amodified phosphate buffered saline excipient (cPBS)) following the sameregimen as the treated animals. The daily doses of active drug andplacebo were administered by momentarily restraining animals in thecages (i.e., employing the squeeze mechanism). Tissues obtained upondeath were prepared and analyzed as described (Giesbert, T. W., et. al.(2002) Emerging Infectious Diseases 8:503-507)

Out of the four treated monkeys there was one survivor that remainsalive to date (male) and two deaths occurring on days 8, 13, and 14 (allfemale). The control male animal died on day 9. From previousexperience, the incidence of mortality for untreated animals in thismodel is ˜100% with death occurring between 7 to 10 days with the meanbeing 8.4 days following infection. The first female that died on day 8started cycling heavily the first day after infection and appeared tosuccumb from bleeding due to cycling that was possibly exacerbated bythe treatment drug. The two other female animals also cycled early inthe disease course however, the time to death was prolonged. The animalthat died on day 13 had low viremia and virus in tissues, (i.e.,appeared to be clearing the infection). The hematocrit on this animalwas 8.4 when it was euthanized. The animal that died on day 14 had aplatelet count of 219 on day 13 but also appeared to be anemic. It hasbeen proposed that that at least one of the treated females and possiblyall three could have been saved with a blood transfusion since it didnot appear that the animals succumbed to normal disease pathogenesisexpected after EBOV infection based on histopathological examination.The lone treated male remains alive to date. This animal clearly hadinfection based on the titer of EBOV antibodies, a significant drop inplatelets from a count of 330 pre-infection to 53 on day 10, postinfarction and a loss of appetite.

An additional experiment was performed in which three male animals wereinfected with Ebola virus and treated with rAcaNAPc2/Pro as describedabove. An additional male was used as a control and did not receiverAcaNAPc2/Pro following infection. In this experiment, one animal in thetreated group was found to have underlying case of shigellosis and wasnot included in the analysis. Out of the two other animals, there wasone who survived and is alive to date and one animal that died on day 10post infection. The control animal died on day 9 post infection. Theanimal that died on day 10 showed signs of recovery and upon necropsyand histopathological examination the internal organs did not resemblethe virus control. The survivor in this study as well as that in theprevious study have high titer anti-Ebola antibodies, indicating thateach animal was exposed to Ebola virus.

In summary, the results from two independent experiments demonstratethat rAcaNAPc2/Pro provided complete protection and a survival benefitto 2 out of 6 (33%), delayed mortality in 3 out of the 6 (50%) and nobenefit in 1 out of 6 (17%) animals infected with Ebola and administeredrAcaNAPc2/pro. Two out of two (100%) of the control animals notreceiving rAcaNAPc2/pro died. Therefore, rNAPc2/Pro has demonstratedsignificant utility in a non-human primate model of Ebola virusinfection that is considered the only model that, at present, isconsidered to reflect the same physiological and clinicalcharacteristics of Ebola virus infection in humans (Geisbert et al.“Evaluation in nonhuman primates of vaccines against Ebola virus”.Emerging Infectious Diseases (2002) 8:503-507; Schou et al, “Marburg andEbola virus infections in laboratory non-human primates: a literaturereview”, Comparative Medicine (2000) 50:108-123).

EXAMPLE 3

Assays for Measuring the Inhibition of the fVIIa/TF Complex by a TestCompound

A. fVIIa/TF fIX Activation Assay

This example measures the ability of a test compound to act as aninhibitor of the catalytic complex of fVIIa/TF, which has a primary rolein initiation of the coagulation response in the ex vivo prothrombintime assay (Example 5). Activation of tritiated Factor IX by therFVIIa/rTF/PLV complex was assessed by determining the respectiveintrinsic inhibition constant, Ki*.

Lyphilized, purified, recombinant human factor VIIa was obtained fromBioPacific, Inc. (Emeryville, Calif.), and reconstituted in HBS (10 mMHEPES, pH 7.5, 150 mM sodium chloride) prior to use. Purified humanFactor X was obtained from Enzyme Research Laboratories, Inc. (SouthBend, Ind.) and Factor Xa (free FXa) was activated and prepared from itas described (Bock, P. E., Craig, P. A., Olson, S. T., and Singh, P.Arch. Biochem. Biophys. 273:375-388 (1989)). Active site-blocked humanFactor Xa (EGR-FXa), which had been irreversibly inactivated withL-Glutamyl-L-glycyl-L-arginyl chloromethylketone, was obtained fromHaematologic Technologies, Inc. (Essex Junction, Vt.). Recombinant humantissue factor (rTF) was produced by a baculovirus-expression system, andpurified to homogeneity by monoclonal antibody affinity chromatography(Corvas International, Inc., San Diego, Calif.).

The purified rTF apoprotein was incorporated into phospholipid vesicles(rTF/PLV), consisting of phosphotidyl choline (75%, w/v) andphosphotidyl serine (25%, w/v) in the presence of detergent, asdescribed by Ruf et al. (Ruf, W., Miles, D. J., Rehemtulla, A., andEdgington, T. S. Methods in Enzymology 222: 209-224 (1993)). Thephospholipids were purchased from Avanti Polar Lipids, (Alabaster,Ala.). The buffer used for all assays was HBSA, HBS containing 0.1%(w/v) bovine serum albumin. All reagents were obtained from SigmaChemical Co. (St. Louis, Mo.), unless otherwise indicated.

The activation of human ³H-Factor IX (FIX) by the rFVIIa/TF complex wasmonitored by measuring the release of the radiolabeled activationpeptide. Purified human fIX was obtained from Haematoligic Technologies,Inc. (Essex Junction, Vt.), and radioactively labeled by reductivetritiation as described (Van Lenten & Ashwell, 1971, JBC 246,1889-1894). The resulting tritiated preparation of FIX had a specificactivity of 194 clotting units/mg as measured in immuno-depleted FIXdeficient plasma (Ortho), and retained 97% of its activity. Theradiospecific activity was 2.7×10⁸ dpm/mg. The Km for the activation of³H-FIX by rFVIIa/rTF/PLV was 25 nM, which was equivalent to the Kmobtained for untreated (unlabeled) FIX.

The assay for Ki* determinations was conducted as follows: rFVIIa andrTF/PLV were combined in a polypropylene test tube, and allowed to forma complex for 10 minutes in HBSA, containing 5 mM CaCl₂. Aliquots ofrFVIIa/rTF/PLV complex were combined in the appropriate polypropylenemicrocentrifuge tubes with EGR-FXa or free FXa, when included, andeither the test compound at various concentrations, after dilution intoHBSA, or HBSA alone (as V_(o) (uninhibited velocity) control). Followingan incubation of 60 minutes at ambient temperature, reactions wereinitiated by the addition of ³H-FIX. The final concentration of thereactants in 420 μl of HBSA was: rFVIIa (50 pM), rTF (2.7 nM), PLV (6.4micromolar), either EGR-FXa or free FXa (300 pM), recombinant AcaNAPc2(5 to 1,500 pM), ³H-FIX (200 nM), and CaCl₂ (5 mM). In addition, abackground control reaction was run that included all of the abovereactants, except rFVIIa.

At specific time points (8, 16, 24, 32 and 40 minutes), 80 μl of thereaction mixture was added to an eppendorf tube that contained an equalvolume of 50 mM EDTA in HBS with 0.5% BSA to stop the reaction; this wasfollowed by the addition of 160 μl of 6% (w/v) trichloroacetic acid. Theprotein was precipitated, and separated from the supernatant bycentrifugation at 16,000×g for 6 minutes at 4° C. The radioactivitycontained in the resulting supernatant was measured by removingtriplicate aliquots that were added to Scintiverse BD (FisherScientific, Fairlawn, N.J.), and quantitated by liquid scintillationcounting. The control rate of activation was determined by linearregression analysis of the soluble counts released over time understeady-state conditions, where less than 5% of the tritiated FIX wasconsumed. The background control (≦1.0% of control velocity) wassubtracted from all samples. Ratios of inhibited pre-equilibrium,steady-state velocities (Vi), containing either test compound to theuninhibited control velocity of rFVIIa/TF alone (V_(o)) were plottedagainst the corresponding concentrations of test compound. These datawere then directly fit to an equation for tight-binding inhibitors(Morrison, J. F., and Walsh, C. T., Adv. Enzymol. 61:201-300 (1988)),from which the apparent equilibrium dissociation inhibitory constantK_(i)* was calculated.

The data for rAcaNAPc2 is presented in Table V which follows Section B,below.

B. Factor VIIa/Tissue Factor Amidolytic Assay

The ability of a test compound to act as an inhibitor of the amidolyticactivity of the fVIIa/TF complex was assessed by determining therespective inhibition constant, Ki*, in the presence and absence ofactive site-blocked human Factor Xa (EGR-fXa).

rFVIIa/rTF amidolytic activity was determined using the chromogenicsubstrate S-2288 (H-D-isoleucyl-L-prolyl-L-arginine-p-nitroaniline),obtained from Kabi Pharmacia Hepar, Inc. (Franklin, Ohio). The substratewas reconstituted in deionized water prior to use. rFVIIa and rTF/PLVwere combined in a polypropylene test tube, and allowed to form acomplex for 10 minutes in HBSA, containing 3 mM CaCl₂. The assay for Ki*determinations was conducted by combining in appropriate wells of aCorning microtiter plate 50 μl of the rFVIIa/rTF/PLV complex, 50 μl ofEGR-FXa and 50 μl of either the test compound at various concentrations,after dilution into HBSA, or HBSA alone (for V_(o) (uninhibitedvelocity) measurement). Following an incubation of 30 minutes at ambienttemperature, the triplicate reactions were initiated by adding 50 μl ofS-2288. The final concentration of reactants in total volume of 200 μlof HBSA was: test compound NAP (0.025 to 25 nM), rFVIIa (750 pM), rTF(3.0 nM), PLV (6.4 micromolar), EGR-FXa (2.5 nM), and S-2288 (3.0 mM, 3×Km).

The amidolytic activity of rFVIIa/rTF/PLV was measured as a linearincrease in the absorbance at 405 nm over 10 minutes (velocity), using aThermo Max® Kinetic Microplate Reader (Molecular Devices, Palo Alto,Calif.), under steady-state conditions, where less than 5% of thesubstrate was consumed. Ratios of inhibited pre-equilibrium,steady-state velocities (Vi), containing test compound to theuninhibited velocity of free fXa alone (V_(o)) were plotted against thecorresponding concentrations of test compound. These data were thendirectly fit to the same equation for tight-binding inhibitors, used inExample 3A, from which the apparent equilibrium dissociation inhibitoryconstant K_(i)* was calculated.

TABLE V Ki* (pM) Amidolytic Assay ³H-FIX Activation Test No Plus No+free Compound Addition EGR-FXa Addition FXa +EGR-FXa AcaNAP2^(a) NI 36± 20 NI 35 ± 5 8.4 ± 1.5 NI = no inhibition ND = not determined ^(a)Madein Pichia pastoris. (See, U.S. Pat. No. 5,866,542.)

EXAMPLE 4

Factor Xa Amidolytic Assay

The ability of a test compound to act as an inhibitor of factor Xacatalytic activity was assessed by determining the test compound-inducedinhibition of amidolytic activity catalyzed by the human enzyme, asrepresented by Ki* values.

The buffer used for all assays was HBSA (10 mM HEPES, pH 7.5, 150 mMsodium chloride, 0.1% bovine serum albumin). All reagents were fromSigma Chemical Co. (St. Louis, Mo.), unless otherwise indicated.

The assay was conducted by combining in appropriate wells of a Corningmicrotiter plate, 50 microliters of HBSA, 50 microliters of the testcompound diluted (0.025 to 35 nM) in HBSA (or HBSA alone for uninhibitedvelocity measurement), and 50 microliters of the Factor Xa enzymediluted in HBSA (prepared from purified human Factor X obtained fromEnzyme Research Laboratories (South Bend, Ind.) according to the methoddescribed by Bock, P. E. et al., Archives of Biocyhem, Biophys. 273: 375(1989). The enzyme was diluted into HBSA prior to the assay in which thefinal concentration was 0.5 nM). Following a 30 minute incubation atambient temperature, 50 microliters of the substrate S2765(N-alpha-benzyloxycarbonyl-D-argininyl-L-glycyl-L-arginine-p-nitroanilidedihydrochloride, obtained from Kabi Diagnostica (or Kabi Pharmacia HeparInc., Franklin, Ohio) and made up in deionized water followed bydilution in HBSA prior to the assay) was added to the wells yielding afinal total volume of 200 microliters and a final concentration of 250micromolar (about 5-times Km). The initial velocity of chromogenicsubstrate hydrolysis was measured by the change in absorbance at 405 nmusing a Thermo Max® Kinetic Microplate Reader (Molecular Devices, PaloAlto, Calif.) over a 5 minute period in which less than 5% of the addedsubstrate was utilized.

Ratios of inhibited pre-equilibrium, steady-state velocities containingtest compound (Vi) to the uninhibited velocity of free fXa alone (V_(o))were plotted against the corresponding concentrations of test compound.These data were then directly fit to an equation for tight-bindinginhibitors (Morrison, J. F., and Walsh, C. T., Adv. Enzymol. 61:201-300(1988)), from which the apparent equilibrium dissociation inhibitoryconstant K_(i)* was calculated.

Table VI below gives the Ki* values for the test compound AcaNAPc2 (SEQ.ID. NO. 59). The data show that AcaNAPc2 did not effectively inhibit FXaamidolytic activity.

TABLE VI Compound Ki* (pM) AcaNAPc2^(a) NI* *NI = no inhibition; amaximum of 15% Inhibition was observed up to 1 μM. ^(a)Made in Pichiapastoris. (See, U.S. Pat. No. 5,866,542.)

EXAMPLE 5

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT)Assays

The ex vivo anticoagulant effects of a test compound was evaluated bymeasuring the prolongation of the activated partial thromboplastin time(aPTT) and prothrombin time (PT) over a broad concentration range of thetest compound.

Fresh frozen pooled normal citrated human plasma was obtained fromGeorge King Biomedical, Overland Park, Kans. Respective measurements ofaPTT and PT were made using the Coag-A-Mate RA4 automated coagulometer(General Diagnostics, Organon Technica, Oklahoma City, Okla.) using theAutomated aPTT PlatelinO L reagent (Organon Technica, Durham, N.C.) andSimplastin® Excel (Organon Technica, Durham, N.C.) respectively, asinitiators of clotting according to the manufacturer's instructions.

The assay was conducted by making a series of dilutions of test compoundin rapidly thawed plasma followed by adding 200 microliters or 100microliters to the wells of the assay carousel for the aPTT or PTmeasurements, respectively. Alternatively, the test compound wasserially diluted into HBSA and 10 μl of each dilution were added to 100μl of normal human plasma in the wells of the Coag-A-Mate assaycarousel.

Concentrations of test compound were plotted against clotting time, anda doubling time concentration was calculated, i.e., a specifiedconcentration of test compound that doubled the control clotting time ofeither of the PT or the aPTT. The control clotting times of NHP in thePT and APTT were 12.1 seconds and 28.5 seconds, respectively.

Table VII below shows the ex vivo anticoagulant effects of AcaNAPc2(SEQ. ID. NO. 3), represented by the concentration of that doubled(doubling concentration) the control clotting time of normal humanplasma in the respective PT and APTT clotting assays relative to acontrol assay where no test compound was present. The data show theactivity of the test compounds as an anticoagulant of clotting humanplasma.

TABLE VII Doubling Doubling Concentration (nM) Concentration (nM)Compound in the PT in the aPTT AcaNApc2^(a) 15 ± 1 105 ± 11 ^(a)Made inPichia pastoris. (See, U.S. Pat. No. 5,866,542.)

EXAMPLE 6

Prothrombinase Inhibition Assay

The ability of a test compound to act as an inhibitor of the activationof prothrombin by Factor Xa that has been assembled into a physiologicprothrombinase complex was assessed by determining the respectiveinhibition constant, Ki*.

Prothrombinase activity was measured using a coupled amidolytic assay,where a preformed complex of human FXa, human Factor Va (FVa), andphospholipid vesicles first activates human prothrombin to thrombin. Theamidolytic activity of the generated thrombin is measured simultaneouslyusing a chromogenic substrate. Purified human FVa was obtained fromHaematologic Technologies, Inc. (Essex Junction, Vt.). Purified humanprothrombin was purchased from Celsus Laboratories, Inc. (Cincinnati,Ohio). The chromogenic substrate Pefachrome t-PA(CH₃SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroanilide) fromPentapharm Ltd (Basel, Switzerland) was purchased from Centerchem, Inc.(Tarrytown, N.Y.). The substrate was reconstituted in deionized waterprior to use. Phospholipid vesicles were made, consisting ofphosphotidyl choline (67%, w/v), phosphatidyl glycerol (16%, w/v),phosphatidyl ethanolamine (10%, w/v), and phosphatidyl serine (7% w/v)in the presence of detergent, as described by Ruf et al. (Ruf, W.,Miles, D. J., Rehemtulla, A., and Edgington, T. S. Methods in Enzymology222: 209-224 (1993)). The phospholipids were purchased from Avanti PolarLipids, (Alabaster, Ala.).

The prothrombinase complex was formed in a polypropylene test tube bycombining FVa, FXa, and phospholipid vesicles (PLV) in HBSA containing 3mM CaCl₂ for 10 minutes. In appropriate wells of a microtiter plate, 50μl of test compound diluted in HBSA, or HBSA alone (for V_(o)(uninhibited velocity) measurement). Following an incubation of 30minutes at room temperature, the triplicate reactions were initiated bythe addition of a substrate solution, containing human prothrombin andthe chromogenic substrate for thrombin, Pefachrome tPA. The finalconcentration of reactants in a total volume of 150 μl of HBSA was: testcompound (0.025 to 25 nM), FXa (250 μM), PLV (5 μM), prothrombin (250nM), Pefachrome tPA (250 μM, 5× Km), and CaCl₂ (3 mM).

The prothrombinase activity of fXa was measured as an increase in theabsorbance at 405 nm over 10 minutes (velocity), exactly as described inExample 4, under steady-state conditions. The absorbance increase wassigmoidal over time, reflecting the coupled reactions of the activationof prothrombin by the FXa-containing prothrombinase complex, and thesubsequent hydrolysis of Pefachrome tPA by the generated thrombin. Thedata from each well of a triplicate were combined and fit byreiterative, linear least squares regression analysis, as a function ofabsorbance versus time², as described (Carson, S. D. Comput. Prog.Biomed. 19: 151-157 (1985)) to determine the initial velocity (V_(i)) ofprothrombin activation. Ratios of inhibited steady-state initialvelocities containing test compound (V_(i)) to the uninhibited velocityof prothrombinase fXa alone (V_(o)) were plotted against thecorresponding concentrations of test compound. These data were directlyfit to the equation for tight-binding inhibitors, as in Example 4 above,and the apparent equilibrium dissociation inhibitory constant K_(i)* wascalculated.

Table VIII below gives the dissociation inhibitor constant (Ki*) ofrecombinant AcaNAPc2 (SEQ. ID. NO. 59) (made in Pichia pastoris) againsthe activation of prothrombin by human fXa incorporated into aprothrombinase complex.

TABLE VIII Compound Ki* (pM) AcaNAPc2^(a) 2.385 ± 283 ^(a)Made in Pichiapastoris. (See, U.S. Pat. No. 5,866,542.)

The data presented in Examples 4, 5 and 6 suggest that AcaNAPc2 appearsto be interacting with FXa in a way that only perturbs themacromolecular interactions of this enzyme with either the substrateand/or cofactor (Factor Va), while not directly inhibiting the catalyticturnover of the peptidyl substrate.

EXAMPLE 7

In vitro Enzyme Assays for Active Specificity Determination

The ability of a test compound to act as a selective inhibitor of fXacatalytic activity or TF/VIIa activity was assessed by determiningwhether the test compound would inhibit other enzymes in an assay at aconcentration that was 100-fold higher than the concentration of thefollowing related serine proteases: thrombin, Factor Xa, Factor XIa,Factor XIIa, kallikrein, activated protein C, plasmin, recombinanttissue plasminogen activator (rt-PA), urokinase, chymotrypsin, trypsin.

A. General Protocol for Enzyme Inhibition Assays

The buffer used for all assays was HBSA (see, Example 4). All substrateswere reconstituted in dionized water, followed by dilution into HBSAprior to the assay. The amidolytic assay for determining the specificityof inhibition of serine proteases was conducted by combining inappropriate wells of a Corning microtiter plate, 50 μl of HBSA, 50 μl oftest compound at a specified concentration diluted in HBSA, or HBSAalone (uninhibited control velocity, V_(o)), and 50 μl of a specifiedenzyme (see specific enzymes below). Following a 30 minute incubation atambient temperature, 50 μl of substrate were added to triplicate wells.The final concentration of reactants in a total volume of 200 μl of HBSAwas: test compound (75 nM), enzyme (750 pM), and chromogenic substrate(as indicated below). The initial velocity of chromogenic substratehydrolysis was measured as a change in absorbance at 405 nm over a 5minute period, in which less than 5% of the added substrate washydrolyzed. The velocities of test samples, containing test compound(V_(i)) were then expressed as a percent of the uninhibited controlvelocity (V_(o)) by the following formula: V_(i)/V_(o)×100, for each ofthe enzymes.

B. Specific Enzyme Assays

i. Thrombin Assay

Thrombin catalytic activity was determined using the chromogenicsubstrate Pefachrome t-PA(CH₃SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroaniline, obtainedfrom Pentapharm Ltd., Basel, Switzerland). The final concentration ofPefachrom t-PA was 250 μM (about 5-times Km). Purified humanalpha-thrombin was obtained from Enzyme Research Laboratories, Inc.(South Bend, Ind.).

ii. Factor Xa Assay

Factor Xa catalytic activity was determined using the chromogenicsubstrate S-2765(N-benzyloxycarbonyl-D-arginine-L-glycine-L-arginine-p-nitroaniline),obtained from Kabi Pharmacia Hepar, Inc. (Franklin, Ohio). Allsubstrates were reconstituted in deionized water prior to use. The finalconcentration of S-2765 was 250 μM (about 5-times Km). Purified humanFactor X was obtained from Enzyme Research Laboratories, Inc. (SouthBend, Ind.) and Factor Xa (FXa) was activated and prepared from it asdescribed (Bock, P. E., Craig, P. A., Olson, S. T., and Singh, P. Arch.Biochem. Biophys. 273:375-388 (1989)).

iii. Factor XIa Assay

Factor FXIa catalytic activity was determined using the chromogenicsubstrate S-2366 (L-Pryoglutamyl-L-prolyl-L-arginine-p-nitroaniline,obtained from Kabi Pharmacia Hepar, Franklin, Ohio). The finalconcentration of S-2366 was 750 μM. Purified human FXIa was obtainedfrom Enzyme Research Laboratories, Inc. (South Bend, Ind.).

iv. Factor XIIa Assay

Factor FXIIa catalytic activity was determined using the chromogenicsubstrate Spectrozyme FXIIa (H-D-CHT-L-glycyl-L-arginine-p-nitroaniline,(obtained from American Diagnostica, Greenwich, Conn.). The finalconcentration of Spectrozyme FXIIa was 100 μM. Purified human FXIIa wasobtained from Enzyme Research Laboratories, Inc. (South Bend, Ind.).

v. Kallikrein Assay

Kallikrein catalytic activity was determined using the chromogenicsubstrate S-2302 (H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline,obtained from Kabi Pharmacia Hepar, Franklin, Ohio). The finalconcentration of S-2302 was 400 μM. Purified human kallikrein wasobtained from Enzyme Research Laboratories, Inc. (South Bend, Ind.).

vi. Activated Protein C (aPC)

Activated Protein C catalytic activity was determined using thechromogenic substrate Spectrozyme PCa(H-D-lysyl-(-Cbo)-L-prolyl-L-arginine-p-nitroaniline) were obtained fromAmerican Diagnostica Inc. (Greenwich, Conn.). The final concentrationwas 400 μM (about 4-times Km). Purified human aPC was obtained fromHematologic Technologies, Inc. (Essex Junction, Vt.).

vii. Plasmin Assay

Plasmin catalytic activity was determined using the chromogenicsubstrate S-2366 (L-Pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline,obtained from Kabi Pharmacia Hepar, Franklin, Ohio). The finalconcentration of S-2366 was 300 μM (about 4-times Km). Purified humanplasmin was obtained from Enzyme Research Laboratories, Inc. (SouthBend, Ind.).

viii. Recombinant Tissue Plasminogen Activator (rt-PA)

rt-PA catalytic activity was determined using the substrate, Pefachromet-PA (CH₃SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroaniline,obtained from Pentapharm Ltd., Basel, Switzerland). The finalconcentration was 500 μM (about 3-times Km). Human rt-PA (Activase®) wasobtained from Genentech, Inc. (So. San Francisco, Calif.).

ix. Urokinase

Urokinase catalytic activity was determined using the substrate S-2444(L-Proglutamyl-L-glycyl-L-arginine-p-nitroaniline, obtained from KabiPharmacia Hepar, Franklin, Ohio). The final concentration of S-2444 was150 μM (about 7-times Km). Human urokinase (Abbokinase®), purified fromcultured human kidney cells, was obtained from Abbott Laboratories(North Chicago, Ill.).

x. Chymotrypsin

Chymotrypsin catalytic activity was determined using the chromogenicsubstrate, S-2586(Methoxy-succinyl-L-argininyl-L-prolyl-L-tyrosine-p-nitroaniline, whichwas obtained from Kabi Pharmacia Hepar, Franklin, Ohio). The finalconcentration of S-2586 was 100 μM (about 8-times Km). Purified(3×-crystallized; CDI) bovine pancreatic-chymotrypsin was obtained fromWorthington Biochemical Corp. (Freehold, N.J.).

xi. Trypsin

Trypsin catalytic activity was determined using the chromogenicsubstrate S-2222 (N-benzoyl-L-isoleucyl-L-glutamyl-(-methylester)-L-arginine-p-nitroaniline, which was obtained from Kabi PharmaciaHepar, Franklin, Ohio). The final concentration of S-2222 was 300 μM(about 5-times Km). Purified human pancreatic trypsin was obtained fromScripps Laboratories (San Diego, Calif.).

xii. Results

Table IX lists the inhibitory effect of recombinant AcaNAPc2 (SEQ. ID.NO. 3) expressed in Pichis pastoris, on the amidolytic activity of 11selected serine proteases. Inhibition is expressed as percent of controlvelocity. These data demonstrate that AcaNAPc2 possesses a high degreeof specificity, and is not a specific inhibitor of FXa.

TABLE IX % Control Velocity + Enzyme AcaNAPc2^(a) FXa 84 ± 3 FIIa 99 ± 3FXIa 103 ± 4  FXIIa 97 ± 1 kallikrein 101 ± 1  aPC 97 ± 3 plasmin 107 ±9  r-tPA 96 ± 2 urokinase 97 ± 1 chymotrypsin 99 ± 0 trypsin 93 ± 4^(a)Made in Pichia pastoris. (See, U.S. Pat. No. 5,866,542.)

1. A method of treating a mammal having a viral hemorrhagic fever whichcomprises administering to said mammal an effective amount of aNematode-Extracted Anticoagulant Protein (“NAP”) having FactorVIIa/tissue factor inhibitory activity (“fVIIa/TF”), wherein said NAPcomprises one or more NAP domains, wherein each NAP domain includes thesequence:Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10,wherein (a) A1 is an amino acid sequence of 7 to 8 amino acid residues;(b) A2 is an amino acid sequence of 3 to 5 amino acid residues; (c) A3is an amino acid sequence of 3 amino acid residues; (d) A4 is an aminoacid sequence of 6 to 19 amino acid residues; (e) A5 is an amino acidsequence of 3 to 4 amino acid residues; (f) A6 is an amino acid sequenceof 3 to 5 amino acid residues; (g) A7 is an amino acid; (h) A8 is anamino acid sequence of 11 to 12 amino acid residues; (i) A9 is an aminoacid sequence of 5 to 7 amino acid residues; and (j) A10 is an aminoacid sequence of 5 to 25 amino acid residues.
 2. A method according toclaim 1 wherein said virus is selected from the group consisting ofFiloviridae, Arenaviridae, Bunyaviridae and Flaviridae.
 3. A methodaccording to claim 2 wherein said virus is from the family Filoviridae.4. A method according to claim 3 wherein said NAP is AcaNAPc2 (SEQ IDNO:3) and said virus is Marburg virus.
 5. A method according to claim 3wherein said NAP is AcaNAPc2/proline (SEQ ID NO: 4) and said virus isMarburg virus.
 6. A method of treating a coagulopathy in a mammal with aviral hemorrhagic fever which comprises administering to said mammal aneffective amount of a Nematode-Extracted Anticoagulant Protein (“NAP”)having Factor VIIa/tissue factor inhibitory activity (“fVIIa/TF”),wherein said NAP comprises one or more NAP domains, wherein each NAPdomain includes the sequence:Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10,wherein (a) A1 is an amino acid sequence of 7 to 8 amino acid residues;(b) A2 is an amino acid sequence of 3 to 5 amino acid residues; (c) A3is an amino acid sequence of 3 amino acid residues; (d) A4 is an aminoacid sequence of 6 to 19 amino acid residues; (e) A5 is an amino acidsequence of 3 to 4 amino acid residues; (f) A6 is an amino acid sequenceof 3 to 5 amino acid residues; (g) A7 is an amino acid; (h) A8 is anamino acid sequence of 11 to 12 amino acid residues; (i) A9 is an aminoacid sequence of 5 to 7 amino acid residues; and (j) A10 is an aminoacid sequence of 5 to 25 amino acid residues.
 7. A method according toclaim 6 wherein said virus is selected from the group consisting ofFiloviridae, Arenaviridae, Bunyaviridae and Flaviridae.
 8. A methodaccording to claim 7 wherein said virus is from the family Filoviridae.9. A method according to claim 8 wherein said NAP is AcaNAPc2 (SEQ IDNO:3) and said virus is Marburg virus.
 10. A method according to claim 8wherein said NAP is AcaNAPc2/proline (SEQ ID NO:4) and said virus isMarburg virus.
 11. A method of treating an inflammatory response in amammal with a viral hemorrhagic fever which comprises administering tosaid mammal an effective amount of a Nematode-Extracted AnticoagulantProtein (“NAP”) having Factor VIIa/tissue factor inhibitory activity(“fVIIa/TF”), wherein said NAP comprises one or more NAP domains,wherein each NAP domain includes the sequence:Cys-A1-Cys-A2-Cys-A3-Cys-A4-cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-cys-A10,wherein (a) A1 is an amino acid sequence of 7 to 8 amino acid residues;(b) A2 is an amino acid sequence of 3 to 5 amino acid residues; (c) A3is an amino acid sequence of 3 amino acid residues; (d) A4 is an aminoacid sequence of 6 to 19 amino acid residues; (e) A5 is an amino acidsequence of 3 to 4 amino acid residues; (f) A6 is an amino acid sequenceof 3 to 5 amino acid residues; (g) A7 is an amino acid; (h) A8 is anamino acid sequence of 11 to 12 amino acid residues; (i) A9 is an aminoacid sequence of 5 to 7 amino acid residues; and (j) A10 is an aminoacid sequence of 5 to 25 amino acid residues.
 12. A method according toclaim 11, wherein said virus is selected from the group consisting ofFiloviridae, Arenaviridae, Bunyaviridae and Flaviridae.
 13. A methodaccording to claim 12 wherein said virus is from the family Filoviridae.14. A method according to claim 13 wherein said NAP is AcaNAPc2 (SEQ IDNO:3) and said virus is Marburg virus.
 15. A method according to claim13 wherein said NAP is AcaNAPc2/proline (SEQ ID NO:4) and said virus isMarburg virus.